Protein synthesis in humans and other Eukarya begins with transcription of the encoding gene. The first step in this process is the binding of the TATA Binding Protein (TBP) to the 'TATA box' sequence within the gene promoter. Our hypothesis is that the unusual structural, energetic and kinetic characteristics of TBP-TATA complexes and their influence on the assembly of the higher order complexes are critical to effective initiation and regulation of transcription. Key issues relating to the complexity and specificity of these binary complexes are addressed in this proposal, with important biological implications for the rate of formation of the transcription pre-initiation complex. The TBP-TATA complex nucleates assembly of Iranscription Factors (TF) IIA and lIB. Together with TBP, TFIIA and TFIIB constitute the minimal ensemble of proteins that can specifically recruit the requisite enzyme, RNA polymerase II, to a promoter. They are also targets for regulatory proteins. Abnormalities in these molecular events in the early stages of pre-initiation complex formation have been implicated in myriad human diseases. This link between transcription and disease provides an opportunity for targeted intervention. Despite their key roles, quantitative molecular mechanisms have not been established for the assembly interactions of the higher order complexes incorporating TFIIA and TFIIB. The proposed work will characterize the time-dependent thermodynamic and structural changes that compose these TBP-nucleated interactions, the essential first step in developing a quantitative predictive model for the initiation and regulation of transcription. The proposed correlation of these biophysical parameters with the corresponding in vivo transcription activity will give needed insight into the rules governing the transcriptional process. While most studies to date have utilized TBP from yeast, the proposed research includes a detailed characterization of the behavior of human TBP. These studies apply an ensemble of quantitative biophysical approaches including time-resolved spectroscopies that allow the integration of conformational, thermodynamic and kinetic characteristics into a comprehensive model of the structure, function and biology of TBP-nucteated complexes. The long-term goal of our research program is to understand the physical-chemical mechanisms by which eukaryotic gene transcription is initiated and regulated.